Chimeric selectins as simultaneous blocking agents for component selectin function

ABSTRACT

Chimeric peptides or polypeptides that combine ligand binding portions from within the lectin and EGF domains of two different selectins are disclosed. The peptides or polypeptides can be constructed solely of the indicated portions of lectin or EGF domains or they can include portions of any of the remaining domains (SCR, transmembrane or cytoplasmic), or the entire extracellular portion, of a generic selectin molecule. The peptides or polypeptides also can be joined to a carrier protein (e.g., a soluble portion of an immunoglobulin molecule) to increase the serum half-life of the therapeutic agent. The chimeric polypeptides can be used as therapeutic agents to antagonize selectin function. They are also useful for screening for agents that are simultaneously antagonists of the function of lectin and EGF domains of different selectins.

GOVERNMENT RIGHTS

Part of the work leading to this invention was made with United States Government funds. Therefore, the U.S. Government has certain rights in this invention.

RELATED APPLICATIONS

This application is a continuation of United States application Ser. No. 08/008,459, filed Jan. 25, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates to the selectin family of receptor adhesion molecules and particularly to agents interfering with selectin function.

BACKGROUND OF THE INVENTION

The ability of leukocytes to leave the circulation and to migrate into tissues is a critical feature of the immune response. Normally, the infiltrating leukocytes phagocytize invading organisms or dead or damaged cells. However, in pathologic inflammation, infiltrating leukocytes can cause serious and sometimes deadly damage. Leukocyte-mediated inflammation is implicated in a number of human clinical manifestations, including the adult respiratory distress syndrome, multi-organ failure and reperfusion injury.

The selectin family of receptor adhesion molecules mediates the initial interactions of leukocytes with endothelium (Springer, Nature 346:425-434 (1990); Lasky, Science 258:964-969 (1992)). L-selectin (also known as LAM-1) is expressed on the surface of most classes of leukocytes (Griffin et al., J. Immunol. 145:576-584 (1990); Tedder et al., J. Immunol. 144:532-540 (1990)) and mediates the binding of lymphocytes to high endothelial venules (HEV) of lymph nodes and to activated endothelium. P-selectin (also called PADGEM, CD62 or GMP-140) is expressed by activated platelets and endothelial cells (Johnston et al., Cell 56:1033 (1989); Hsu-Lin et al., J. Biol. Chem. 259:9121 (1984)) and mediates adhesion between myeloid cells and activated endothelium or activated platelets (Gallatin et al, Nature 304:30-34 (1983); Spertini et al, J. Immunol. 147:2565-2573 (1991); Larsen et al, Cell 59:305-312 (1989); Geng et al, Nature 343:757-760 (1990)). Another member of the family E-selectin (also known as ELAM-1) is expressed by activated endothelial cells (Bevilacqua et al., Science 243:1160 (1989); Bevilacqua et al., Proc. Nat'l Acad. Sci. USA 84:9238 (1987)) and partially mediates the binding of neutrophils to endothelium at sites of inflammation (Bevilacqua et al., Proc. Nat'l Acad. Sci. USA 84:9238 (1987)). All selectins are derived from evolutionarily related genes (Collins et al., J. Biol. Chem. 266:2466-2478 (1991); Johnston et al., J. Biol. Chem. 34:21381-21385 (1990); Ord et al., J. Biol. Chem. 265:7760-7767 (1990); Watson et al., J. Exp. Med. 172:263-272 (1990)), and are characterized by an NH₂ -terminal, Ca⁺ -dependent lectin domain, an epidermal growth factor (EGF)-like domain followed by multiple short consensus repeat (SCR) domains, a transmembrane region, and a cytoplasmic tail. Although the lectin domains are critical for the binding of specific carbohydrate ligands (Springer, Nature 346:425-434 (1990); Lasky, Science 258:964-969 (1992)), the role of the conserved EGF-like domains is unknown.

It has been proposed that the treatment of a patient suffering from pathologic inflammation with an antagonist to adhesion receptor function can result in the reduction of leukocyte migration to a level manageable by the target endothelial cells and the subsequent dramatic recovery of the patient. Local administration of therapeutic agents can block competitively the adhesive interactions between leukocytes and the endothelium adjacent to an inflamed region. Therapeutic agents can also be administered on a systemic level for the treatment of a patient suffering from disseminated inflammation (Harlan and Liu, eds., Adhesion: Its Role in Inflammatory Disease, W. H. Freeman (in press)).

SUMMARY OF THE INVENTION

We report here that the EGF-like domains of P-selectin and of E-selectin can participate directly in cell adhesion, having ligand binding sites distinct from those of their respective lectin domains. Cell adhesion mediated at least by the P and E-selectins may be complex, involving interactions between the lectin domain and a carbohydrate ligand, and separately, between the EGF-like domain and one or more proteins or other ligands. Our discovery of a difference in at least one of the ligands recognized by the lectin and EGF domains of a given selectin has permitted the preparation of chimeric polypeptides that combine within one molecule the ability to target two or more different selectin ligands and the use of these agents in therapy or in the preparation of additional classes of antagonists to selectin function.

Thus, the invention generally features chimeric peptides or polypeptides that combine ligand binding portions from within the lectin and EGF domains of two different selecting. The peptides or polypeptides can be composed solely of the indicated portions of lectin or EGF domains or they can include portions of any of the remaining domains (SCR, transmembrane or cytoplasmic), or the entire extracellular portion, of a generic selectin molecule. The peptides or polypeptides also can be joined to a carrier protein (e.g., a soluble portion of an immunoglobulin molecule) to increase the serum half-life of the therapeutic agent. In another aspect, the invention features nucleic acid encoding the chimeric polypeptides.

The chimeric polypeptides can be used as therapeutic agents to antagonize selectin function. They are also useful for screening for agents that are simultaneously antagonists of the function of lectin and EGF domains of different selecting.

As used herein, the term "polypeptide" is intended to include shorter molecules, or "peptides." The term "essentially purified" refers to a polypeptide sequence that has been separated or isolated from the environment in which it was prepared or in which it naturally occurs.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, taken in conjunction with the accompanying drawings in which:

FIG. 1 shows the structure and expression of L-selectin/P-selectin chimeric proteins (Lectin, lectin domain; EGF, epidermal growth factor-like domain; SCR, short consensus repeat; TM, transmembrane domain; C, cytoplasmic domain);

FIG. 2a shows binding of HL-60 cells to COS cells transfected with natural and chimeric FIG. 2A: natural P-selectin; FIG. 2B: natural L-selectin; FIG. 2C: chimeric selectin P2L; FIG. 2D: chimeric selectin L2P. (Original magnification, 200x);

FIG. 2E shows mapping of the domain(s) of L2P responsible for adhesion of HL-60 cells (bars represent the means ±SD, and are representative of at least six experiments);

FIG. 3A shows neuraminidase sensitivity for adhesion mediated by the lectin and EGF domains of P-selectin. Horizontal hatching: untreated cells; tight diagonal hatching: neuraminidase treated cells; open diagonal hatching: cells pretreated with C1 mAb.

FIG. 3B shows protease sensitivity and Ca²⁺ requirement for adhesion mediated by the lectin and EGF domains of P-selectin. Horizontal hatching: untreated cells; open: chymotrypsin treated cells; cross-hatching: papain treated cells; open: cells treated with 25 mM EDTA.

FIG. 3C shows an analysis of the binding of the chimera L2P3L. Horizontal hatching: untreated cells; close diagonal hatching: neuraminidase treated cells; open diagonal hatching: cells pretreated with LAM1-3 mAb; open: chymotrypsin treated cells; cross-hatching: papain treated cells; open: cells treated with 25 mM EDTA.

FIGS. 4A-4C show the cDNA nucleotide sequence (SEQ ID NO: 1) encoding L-selectin and also show the amino acid sequence of L-selectin (SEQ ID NO: 2);

FIG. 5A shows the location relative to a restriction enzyme map of the lyam-1 gene of the overlapping genomic fragments from seven clones containing inserts that hybridized with LAM-1 cDNA probes.

FIG. 5B is a restriction enzyme map of the lyam-1 gene.

FIG. 5C shows the exon-intron organization of the lyam-1 gene.

FIGS. 6A, 6B and 6C show the nucleotide sequence of exons II through X of the lyam-1 gene (SEQ ID NOS: 3-11).

FIG. 7A shows the homology between the lectin domain of human L-selectin (residues 52-170 of SEQ ID NO:2) and the lectin domains of : mouse (SEQ ID NO:12), rat (SEQ ID NO:13) and bovine (SEQ ID NO:14) L-selectin; human (SEQ ID NO:15) and bovine (SEQ ID NO:16) E-selectin; and human (SEQ ID NO:17), mouse (SEQ ID NO:18) and bovine (SEQ ID NO:19) P-selectin.

FIG. 7B shows the homology between the EGF domain of human L-selectin (residues 171-206 of SEQ ID NO:2) and the EGF domains of: mouse (SEQ ID NO:20), rat (SEQ ID NO:21) and bovine (SEQ ID NO:22) L-selectin; human (SEQ ID NO:23), mouse (SEQ ID NO:2) and bovine (SEQ ID NO:25) E-selectin; and human (SEQ ID NO:26), mouse (SEQ ID NO:27) and bovine (SEQ ID NO:28) P-selectin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We have examined the role of the lectin and EGF-like domains of the L-, P-, and E-selectins in cell adhesion and have determined that the EGF-like domains of P- and E-selectin play a direct role in ligand recognition and leukocyte adhesion mediated by the respective selectin. This discovery has permitted the preparation of chimeric polypeptides that combine within one therapeutic agent the ability to target two or more different selectin ligands and the use of these agents in therapy or in the preparation of additional classes of antagonists to selectin function.

Determination of the binding specificity of selectin lectin and EGF domains

The functional activities of the lectin and EGF-like domains of L-, P-, and E-selectin in the adhesive events described above were examined using a panel of L-selectin/P-selectin and L-selectin/E-selectin chimeric molecules. Referring to FIG. 1, these hybrid polypeptides were created by exchange of cDNA encoding the lectin, EGF, or both lectin and EGF domains of the desired selectin and expression of the chimeric nucleic acid, as described in the Experimental Procedures section. Many other methods familiar to those of skill in the art can be employed to prepare similar products.

The chimeras conserve the overall polypeptide backbone structure of the selectins, and resulted in minimal changes in the junctions between domains. The functional characterization of these chimeric selectins therefore offered a powerful approach to determining the molecular basis of cell adhesion mediated by these molecules and to preparing diagnostic and therapeutic agents incorporating properties of individual selectins into the same polypeptide.

Expression of the chimeric proteins in COS cells was verified and quantitated using panels of P-, E- and L-selectin domain-specific monoclonal antibodies (mAb) in direct adhesion assays. The chimeric cDNA were subcloned into the pMT-2 vector and used to transiently transfect COS cells as described under Experimental Procedures. Twenty-four hours before analysis, 2.5×10⁴ transfected COS cells were replated into 96 well plates. Expression of domain-specific epitopes by chimeric selectins was assessed using mAb reactive with specific domains and a surface immunofluorescence assay as described (Luscinskas et al, J. Immunol. 149:2163-2171 (1992)).

Referring again to FIG. 1, the individual constructs, containing the indicated domains of P-selectin and L-selectin, reacts only with anti-L-selectin and anti-P-selectin monoclonal antibodies recognizing the specific, indicated domains. These results demonstrate that the chimeric cDNA encoded the expected proteins and that each protein was expressed and readily detected on the surface of COS cells. Note that the LAM1-1 mAb fails to recognize either the L2P or P2L proteins, and therefore defines an epitope composed of residues in both the lectin and EGF domains. Values given are means of absolute optical density (OD) units from which OD units obtained with a control non-binding mAb were subtracted. Experimental values less than the control values are represented as zero. The values presented are typical of those obtained in three experiments. The standard deviations for these fluorescence measurements were less than 15%.

Characterization of the adhesion reactions mediated by the selectin lectin and EGF domains

The HL-60 myelomonocytic cell line expresses ligands for P-selectin (Larsen et al, Cell 59:305-312 (1989); Geng et al, Nature 343:757-760 (1990)) and E-selectin (Bevilacqua et al., Science 243:1160 (1989); Bevilacqua et al., Proc. Nat'l Acad. Sci. USA 84:9238 (1987)) and was therefore used to assess P-selectin and E-selectin function. Referring to FIGS. 2A-2D, HL-60 cells bound at high levels to COS cells expressing P-selectin, but did not bind to COS cells expressing L-selectin. HL-60 cells also bound to COS cells expressing the chimeric protein P2L, in which the lectin domain from P-selectin was substituted for the lectin domain of L-selectin. Therefore, the lectin domain of P-selectin alone, when attached to the EGF-like and other domains from L-selectin, was sufficient to mediate high levels of HL-60 cell adhesion. However, the lectin domain of P-selectin alone was not solely responsible for all HL-60 binding, because HL-60 cells also bound to COS cells expressing L2P, in which the P-selectin lectin domain was replaced with that of L-selectin.

The domain(s) within L2P responsible for HL-60 adhesion were mapped using additional chimeric selectins. Referring to FIG. 2E, HL-60 cells bound to COS cells expressing L2P3L, which contains only the EGF domain from P-selectin, but did not bind to COS cells expressing L3P, which lacks both the lectin and EGF-like domains of P-selectin but contains the P-selectin SCR domains. Furthermore, binding of HL-60 cells to P3L, which contains both the lectin and EGF domains from P-selectin, was approximately equivalent to that of native P-selectin and significantly (P<0.01; Student's T test) higher than binding of cells to P2L, even though expression of P2L and P3L were approximately equal. These data indicate that both the lectin and EGF-like domains of P-selectin contain ligand binding sites capable of independently mediating HL-60 cell adhesion.

For study of ligand binding sites in E-selectin, four L-selectin/E-selectin chimeric molecules were constructed. L2E has the lectin of L-selectin and the remainder of E-selectin; E2L is the precise converse. L5E has the amino terminal 88 amino acids of L-selectin (i.e., 74% of the lectin domain) and E5L is the precise converse. Binding of HL-60 cells to COS cells expressing these chimeras was assessed as before. The constructs containing only a portion of a specific lectin domain exhibited zero binding, while the binding to chimeras containing lectin and EGF domains from different selectins was reduced by about a half. Therefore, the overall pattern of binding was similar to the results with L-selectin/P-selectin chimeras.

Determination of the influence of EGF-domains on the adhesive activity or specificity of the lectin domains

Adhesion mediated by P-selectin involves sialic acid-bearing carbohydrates, possibly including the sLe^(x) and/or related structures, as well as protein determinants (Larsen et al, Cell 63:467-474 (1990); Zhou et al, J. Cell Biol. 115:557-564 (1991); Moore et al, J. Cell. Biol. 118:445-456 (1992); Moore et al, J. Cell Biol. 112:491-499 (1991); Polley et al, Proc. Natl. Acad. Sci. USA 88:6224-6228 (1991); Larsen et al, J. Biol. Chem. 267:11104-11110 (1992)). Therefore, the effects of neuraminidase and protease treatment of HL-60 cells on binding to COS cells expressing either P-selectin, P3L, or P2L was examined.

Removal of sialic acid residues from HL-60 cells significantly (P<0.01) but incompletely reduced adhesion of HL-60 cells to COS cells expressing native P-selectin or P3L (FIG. 3A). In contrast, removal of sialic acid residues from HL-60 cells nearly completely eliminated adhesion of HL-60 cells to P2L (FIG. 3A). In addition, binding to the E-selectin/L-selectin construct E2L was partially inhibited by pretreatment of the HL-60 cells with CSLEX1 mAb directed against sLex. Similarly, pretreatment of COS cells with mAb directed against the lectin domain of P-selectin significantly (P<0.005), but only partially, inhibited the adhesion of HL-60 cells to P-selectin or P3L, but abolished adhesion to P2L (FIG. 3A).

Treatment of HL-60 cells with either chymotrypsin or papain eliminated >90% of binding to COS cells expressing P-selectin, P3L, or P2L (FIG. 3B). In addition, 2.5 mM EGTA abolished adhesion (FIG. 3B). These results confirm the importance of the lectin domain in adhesion, and offer independent evidence that the EGF domain of P-selectin also plays a direct role in adhesion, recognizing a protein ligand.

A similar analysis was performed on L2P3L to characterize adhesion mediated by the EGF-like domain of P-selectin. Adhesion of HL-60 cells to COS cells expressing L2P3L was also reduced, but not eliminated, by neuraminidase treatment of the HL-60 cells (FIG. 3C). MAb directed against the L-selectin lectin domain present within L2P3L also significantly (P<0.01) inhibited adhesion. These results suggest that recognition of specific carbohydrate structures on HL-60 cells by the lectin domain of L-selectin, which alone was not sufficient for cell adhesion, contributes to leukocyte adhesion in this system when this interaction is supported by the adhesive activity mediated by the EGF domain of P-selectin. Consistent with this hypothesis, rare (˜1 plate) rosettes of HL-60 cells were occasionally observed on COS cells transfected with L-selectin (data not shown), and a soluble L-selectin/IgG fusion protein has a measurable affinity for purified, immobilized sLe^(x) (Foxall et al, J. Cell Biol. 117:895-902 (1992)). Adhesion of HL-60 cells to L2P3L was eliminated by EGTA, indicating that adhesion mediated by the EGF-like domain is also Ca²⁺ -dependent, and was significantly (P<0.01) reduced by protease treatment (FIG. 3C).

Determination of the influence of EGF domains on the adhesive activity or specificity of the lectin domains

In order to determine if the EGF-like domains of selectins have any influence on the adhesive activity or specificity of the lectin domain, stable transfectants of the 300.19 murine pre-B cell line expressing L-selectin, P-selectin, L2P or L2P3L were generated. These cells were tested for binding to lymph node HEV using the standard in vitro frozen section assay (Stamper Jr. et al, J. Exp. Med. 144:828-833 (1976)). Transfectants expressing P-selectin did not bind to HEV, whereas transfectants expressing L-selectin, L2P or L2P3L bound to HEV equivalently and at high levels (Table I).

                  TABLE I                                                          ______________________________________                                         Adhesion of cell lines to lymph node HEV is mediated by                        the lectin domain of L-selectin.                                                       LAM1-3                                                                 cDNA    mAb      expt 1     expt 2   expt 3                                    ______________________________________                                         none    -        0.047 ± 0.02                                                                            0.12 ± 0.025                                                                        <0.01                                             +        <0.01      ND       ND                                        L-selectin                                                                             -        3.43 ± 0.42                                                                            6.85 ± 0.45                                                                          2.44 ± 0.51                                    +        0.05 ± 0.2                                                                             ND       <0.01                                     L2P     -        3.71 ± 0.26                                                                            6.64 ± 0.43                                                                          4.40 ± 0.3                                     +        0.074 ± 0.03                                                                           ND       <0.01                                     L2P3L   -        5.71 ± 0.33                                                                            6.85 ± 0.18                                                                          3.94 ± 0.27                                    +        0.08 ± 0.03                                                                            ND       <0.01                                     P-selectin                                                                             -        <0.01      <0.01    ND                                        ______________________________________                                    

The lectin domain of L-selectin alone was therefore capable of mediating binding to HEV when attached to the EGF-like and other domains of P-selectin. The L2P and L2P3L chimeric selectins can therefore bind to different types of cells which express ligand(s) for either L- or P-selectin, reflecting the specificities of both of the parent selectins from which they are constructed. This acquisition of novel adhesive properties as a result of the exchange of lectin or EGF-like domains between selectins reinforces our hypothesis that the EGF-like domain of P-selectin can mediate cell adhesion. In addition, these data directly demonstrate the functional independence of the lectin and EGF-like domains, and therefore argue against a role for the EGF-like domain in determining the carbohydrate specificity of the lectin domain.

Structure of the L-selectin gene

As disclosed in U.S. patent application Ser. No. 07/983,606, pending, cDNA encoding L-selectin has been cloned and the sequence determined, as shown in FIGS. 4A, 4B and 4C (SEQ ID NO: 1). The cDNA encodes a protein of 372 amino acids (SEQ ID NO: 2).

The structure of the lyam-1 gene, which encodes the LAM-1 protein, was determined by isolating overlapping genomic DNA clones that hybridized with a LAM-1 cDNA probe. The lyam-1 gene spans greater than 30 kb of DNA and is composed of at least 10 exons. The 5' end of the LAM-1 mRNA was mapped by primer extension analysis, revealing a single initiation region for transcription. Exons II through X contain translated sequences; exon II (SEQ ID NO: 3) encodes the translation initiation codon, residue 14 shown in FIG. 4B; exon III (SEQ ID NO: 4) encodes the leader peptide domain, residues 15-51; exon IV (SEQ ID NO: 5) encodes the lectin-like domain, residues 52-170; exon V (SEQ ID NO: 6) encodes the epidermal growth factor-like domain, residues 171-206; exons VI (SEQ ID NO: 7) and VII (SEQ ID NO: 8) encode the short consensus repeat unit domains, residues 207-269 and 270-331; exon VIII (SEQ ID NO: 9) encodes the transmembrane region, residues 332-373; exon IX (SEQ ID NO: 10) encodes seven amino acids containing a potential phosphorylation site, residues 374-380; and exon X (SEQ ID NO: 11) encodes the five remaining amino acids of the cytoplasmic tail and the long 3' untranslated region.

The pLAM-1 cDNA was labeled with ³² P and used as a probe to isolate hybridizing DNAs from a human leukocyte genomic DNA library. Approximately 1×10⁶ plaques were screened, and 13 plaques that hybridized with the cDNA probe were identified and isolated. Seven of these clones were found to contain inserts with unique restriction enzyme maps representing overlapping genomic fragments spanning at least 30 kb. These inserts, LAMG-17, -19, -20, -28, -35, -37, and -47, were further digested and subcloned into plasmids. Detailed restriction maps of these subclones were made and compared to those of intact inserts to determine their correct locations (FIGS. 5A and 5B).

The correctness of the restriction map was verified with Southern blot analysis. DNA isolated from two B cell lines, BL and BJAB, and one T cell line, HSB-2, was digested to completion with Bam HI, Bal II, or Pvu II, size-fractionated, and transferred onto nitrocellulose. This filter was probed with the LAM-1 cDNA clone, pLAM-1. All genomic fragments derived from endonuclease digested DNA hybridized with cDNA probe to generate hybridizing bands of the appropriate size.

The pLAM-1 cDNA clone encodes an 85-bp 5' untranslated region. An oligonucleotide homologous with the 5' sequence of the pLAM-1 cDNA was used as a probe for primer extension analysis. This oligonucleotide was hybridized with poly (A⁺) RNA isolated from the human B cell line RAJI, the LAM-1 negative human B cell line Namalwa, the mouse pre-B cell line A20, and yeast tRNA as a control. Complementary DNA was synthesized by extending the primer with reverse transcriptase. The major primer extension product obtained using the human LAM-1 positive B cell line RNA was extended 126 nucleotides beyond the translation initiation site. There was a single cluster of transcription initiation sites for the lyam-1 gene apparent in the reaction with RAJI RNA that was not found with the LAM-1 negative Namalwa RNA. Several primer extension products of size similar to those of the human B cell line RNA were obtained with mouse B cell RNA. Therefore, murine B cells may express an RNA species that cross-hybridizes with the oligonucleotide probe used. No primer extension products were obtained in the yeast tRNA control reactions.

The relationship of the primer extension results to the cloned LAM-1 cDNAs and the most 5' exon of lyam-1 isolated was used to determine the nucleotide sequence of the exon that encodes the translation initiation AUG codon. This exon ends immediately after the site that encodes the translation initiation codon (FIG. 6A) and overlaps precisely with the pLAM-1 cDNA sequence. The length of the cDNA clone obtained by Bowen et al., J. Cell Biol. 109:421-427 (1989) agrees precisely with the primer extension results except for two nucleotides. However, 15 nucleotides before the 5' end of the cDNA the sequence diverges from the genomic sequence at a site homologous with the 3' splice acceptor site consensus sequence. Therefore, it is most likely that this 15-bp region is derived from the exon upstream of this potential splice site. Thus, the primer extension results indicate that exon I would most likely be composed of 15 or fewer base pairs.

A 15-bp oligonucleotide homologous with the 5' nucleotides present in the cDNA clone of Bowen et al., supra but not encoded by exon II, was used to probe the 10 kb of cloned DNAs 5' of exon II; however, specific hybridization was not detected by Southern blot analysis. Under the conditions necessary for hybridization of this oligonucleotide, significant cross-hybridization occurred with λ-DNA, making it difficult to use this oligonucleotide to isolate the first exon from a λ-based genomic library.

These results suggest that the exon which encodes the translation initiation site is the second exon of the lyam-1 gene (FIG. 5). Consistent with this, the 900 bp upstream of exon II did not contain any apparent "TATA" or "CCAAT" sequences frequently found in promoter regions of eukaryotic genes (FIG. 5). Therefore, it is likely that the transcription initiation region and exon I are further than 10 kb upstream from exon II of the lyam-1 gene. S1 nuclease protection analysis was carried out using the 5' region of exon II as a labeled probe for hybridization with poly (A⁺) RNA from RAJI, Namalwa, and A20 cells. Two mRNA species were protected in the RAJI mRNA, while no S1 protection was provided by the other RNAS. The length of these fragments was consistent with differential splicing at the two potential CAG/N splice sites located within the potential splice acceptor site in exon II. It is therefore likely that the transcription initiation region has not been identified.

The majority of the exons were localized by comparison of the restriction enzyme maps of the genomic clones and the pLAM-1 cDNA. In cases where this method did not provide definitive results, subcloned DNA fragments were digested with selected restriction enzymes, electrophoresed through agarose gels, and transferred to nitrocellulose. Fragments containing exons were identified by Southern blot analysis using labeled cDNA or oligonucleotide probes. The exon that encodes the 3' untranslated region of the LAM-1 cDNA was not contained within the 30 kb of isolated DNA fragments. Therefore, a labeled 0.9-kb Dra I fragment containing most of the 3' untranslated region of the pLAM-1 cDNA was used as a probe to identify a homologous 3.2-kb fragment generated by complete Eco RI digestion of genomic DNA. Eco RI-digested genomic DNA fragments of this size were used to make a partial λ-gtll genomic library from which the 3.2-kb Eco RI fragment was cloned. This 3.2-kb fragment did not overlap with the previously isolated genomic DNAs.

The exact boundaries of the exons were determined by nucleotide sequence analysis. From this analysis, nine exons were identified which make up the entire pLAM-1 cDNA. Exon II encodes the translation initiation codon, and exon III encodes the leader domain of the LAM-1 protein (FIG. 5). Each of the lectin-like, epidermal growth factor-like, transmembrane, and short consensus repeat domains was encoded by a separate exon. The smallest exon, IX, is 19 bp in length and may encode a carboxyl-terminal phosphorylation cassette. The last 5 amino acids of the LAM-1 protein and the 3' untranslated region which includes the poly(A) attachment site, AATAAA, are encoded by exon X as shown in FIG. 6C. The nine exons which encode pLAM-1 were split inside codons in all cases, except the junction between exons II and III. In each instance, the consensus sequences of 5' donor splice sites and 3' acceptor splice sites were adhered to. Nucleotide sequence polymorphisms within the coding region were observed between the genomic clones containing exon V that encoded SCR I and the pLAM-1 clone at cDNA nucleotide positions 741 and 747 (A to G), leading to a coding change from Asn to Ser in both cases, and at position 816 (A to G) changing the Glu to a Gly.

Experimental Procedures Construction of Chimeric Selectin cDNAs

Four new restriction endonuclease recognition sites were introduced separately into the pLAM-1 cDNA by polymerase chain reaction (PCR)-based site-directed mutagenesis as follows. Oligonucleotides surrounding and including each new restriction sequence were synthesized in both the sense and antisense directions. PCR was carried out using the new restriction site sense oligonucleotide and an antisense oligonucleotide anchor located in the plasmid near the 3' end of the cDNA. In a separate reaction, the new restriction site antisense oligonucleotide plus a sense oligonucleotide anchor from the plasmid 5' end were used to amplify the 5' end of the LAM-1 cDNA. The individual PCR products from these two reactions were gel purified, subcloned into the pSP65 vector (Promega Biotec, Madison, Wis.), digested with the appropriate restriction enzymes, and ligated together. The nucleotide changes were as follows: (a) GA³⁶¹ ATCC to GGATCC in the lectin domain, creating a BamHI site, corresponding to amino acid number 53 in the mature protein (FIG. 4A); (b) A⁵⁸³ TGCAG to CTGCAG in the EGF domain, creating a PstI site, corresponding to amino acid number 127; (c) GAGG⁸⁸¹ C⁸⁸² C to GAGCTC in the first SCR domain, creating a SacI site, corresponding to amino acid number 164; and (d) GTCAAA¹⁰²³ to GTCAAC in the second SCR domain, creating a HincII site, corresponding to amino acid number 279. The fidelity of the site-directed mutagenesis was confirmed by restriction mapping and sequence analysis of the cDNAs. Each of these sites is naturally present in the PADGEM cDNA (Larsen et al., Cell 59:305-312 (1984)).

Domain Mapping of Binding Sites

The chimeric selectin cDNAs were subcloned into the pMT-2 expression vector (Kaufman et al, J. Mol. Cell. Biol. 9:946-958 (1989)), and COS cells at ˜50% confluency were transiently transfected with 3 μg of the indicated cDNA by the DEAE dextran method. One day after transfection, the COS cells were replated at ˜50% confluency onto 35 mm Petri dishes (assay plates) (Becton-Dickinson, Lincoln Park, N.J.). The following day, HL-60 cells were washed twice in cold RPMI 1640 media (Gibco, Grand Island, N.Y.), resuspended in RPMI 1640 at a final concentration of 3.3×10⁶ cells ml, and 0.6 ml (2×10⁶ cells) were added to assay plates which had been washed twice with cold RPMI 1640 media. The plates were gently rocked for 20 min at 4° C., washed five times, and the number of HL-60 cells bound to individual COS cells were counted on a minimum of 100 COS cells.

Assay for leukocyte attachment to HEV

The cDNAs encoding native L-selectin, L2P and L2P3L were subcloned into the Bam HI site of the pZIPneoSV(X) vector (Cepko et al, Cell 37:1053-1062 (1984)), and were used to transfect the mouse pre-B cell line 300.19. Stable transfectants were selected in medium containing 0.5-1.0 mg/ml G418 (geneticin; Sigma), and cells expressing the proteins were selected by panning with the LAM1-3 mAb (Spertini et al, J. Immunol. 147:942-949 (1991)). To generate P-selectin transfectants, the P-selectin cDNA in the pMT-2 vector was cotransfected with the pSV2neo vector containing the neomycin resistance marker. Rat lymph nodes were obtained from freshly euthanized Lewis rats, snap frozen in isopentane/liquid nitrogen, and stored at -70° C. in isopentane until use. For the HEV assay, 5×10⁶ of each cell type were incubated on three 12 mm sections/ slide at 64 rpm for 25 min at ˜4° C., the excess cells were gently removed, and the slides were placed vertically in ice-cold fixative (PBS/2.4% glutaraldehyde) overnight. The slides were then counterstained with Gill's hematoxylin, overlaid with glycerol gelatin, and cover slips were applied. Each slide was scored for the number of lymphocytes bound/HEV. Between 100-200 HEV were counted for each experiment. Data are presented as mean ± standard deviation of lymphocytes bound/HEV. ND, not determined.

Determination of neuraminidase sensitivity, protease sensitivity and Ca²⁺ requirements for adhesion

COS cells were transfected with the indicated cDNA and replated as described above. HL-60 cells were incubated in RPMI 1640 medium at 37° C. for two hours with either 0.25 U/ml Clostridium perfringens neuraminidase (Type VI, Sigma, St. Louis, Mo.), 50 mg/ml chymotrypsin or papain (Sigma), washed twice, and the assay was performed as described in the FIG. 2 legend. Neuraminidase treatment of the HL-60 cells was effective in eliminating all reactivity with the CSLEX1 anti-sLe^(x) mAb (data not shown). In some groups, COS cells were preincubated with either 5 μg/ml G1 mAb (a gift: of Dr. R. P. McEver) (Geng et al, Nature 343:757-760 (1990)), or ascites fluid containing the LAM1-3 mAb (Spertini et al, J. Immunol. 147:942-949 (1991)) diluted 1:200. To determine the effect of EGTA, both the HL-60 cells and the COS cells were washed in RPMI 1640 containing 2.5 mM EGTA, and this medium was also used for the assay. No detachment of COS cells occurred under these conditions. These results are representative of at least four experiments.

Use

The adhesion of leukocytes to the endothelial wall of blood vessels and their infiltration into the surrounding tissues contributes to inflammation of tissue at the cellular level. Normally, the infiltrating leukocytes phagocytize invading organisms or dead or damaged cells. However, in pathologic inflammation, infiltrating leukocytes can cause serious and sometimes deadly damage. Leukocyte-mediated inflammation is involved in a number of human clinical manifestations, including the adult respiratory distress syndrome, multi-organ failure and reperfusion injury.

Local administration of therapeutic agents antagonistic to the function of the selectins can block competitively the adhesive interactions between leukocytes and the endothelium adjacent to an inflamed region. On a systemic level, treatment of a patient in shock (e.g., from a serious injury) with an antagonist to selectin function can result in the reduction of leukocyte migration and adhesion and the subsequent dramatic recovery of the patient.

Chimeric peptides or polypeptides combining ligand binding portions from within the lectin and EGF domains of two different selectins can be used for therapeutic treatment to interfere in the binding of leukocytes at the site of inflammation. Referring to FIG. 7, the close homology among the lectin and EGF domains of L-, P- and E-selectin can be seen, and the intron/exon organization of the respective genes encoding P- and E-selectin (Collins et al., J. Biol.Chem. 266:2466 (1991); Johnston et al., J. Biol. Chem. 265:21381 (1990)) is the same as that of the lyam-1 gene encoding L-selectin, disclosed herein. These chimeric agents will offer more efficient targeting than using a single function antagonist. Therapeutic agents of the invention can also be used to block platelet or platelet and mononuclear cell aggregation, thus preventing thrombus formation.

The therapeutic peptides or polypeptides can be composed solely of the indicated portions of lectin or EGF domains or they can include portions of any of the remaining domains (SCR, transmembrane or cytoplasmic), or the entire extracellular portion of a generic selectin molecule. The peptides or polypeptides also can be joined to a carrier protein (e.g., a soluble portion of an immunoglobulin molecule) to increase the serum half-life of the therapeutic agent. Immunoglobulin chimera are easily purified through standard immunochemistry procedures, including IgG-binding protein A-Sepharose chromatography. The chimera have the ability to form an immunoglobulin-like dimer with the concomitant higher avidity and serum half-life.

cDNA sequences encoding the chimeric polypeptides can be incorporated into replicable expression vectors and the vectors transfected into an appropriate host to express the encoded polypeptide. Stop codons can be incorporated into the DNA sequences or any other chain termination method can be used to prepare a soluble polypeptide. The expressed polypeptide can be purified by ordinary techniques well known to those of skill in the art, e.g., by affinity chromatographic methods.

Other agents can also be joined to the therapeutic polypeptides to form a useful product. For example, the ligand binding portions of the lectin and EGF domains of two different selectins can be combined with the toxic portion of a cytotoxin to produce a fusion protein. In addition, the selectin chimeric polypeptides may be coupled to a chemotherapeutic drug or drugs which could simultaneously bind to cells expressing their respective ligands, to administer the drug to a site of tissue damage or inflammation to treat, e.g., acute inflammation or vasculitis. Such drugs may include, anti-inflammatory agents or agents that provide regional relief from inflammatory distress. Syndromes, diseases, and conditions that could be treated by these agents would include, but not be limited to, treating inflammation, microbial/parasitic infections, post-reperfusion injury, leukemia, lymphoma, vasculitis, inhibition of the metastatic spread of tumor cells, organ transplantation, or graft rejection. As is well known to those of skill in the art, the fusion proteins can be transcribed from a hybrid cDNA molecule, or the agent may be covalently bonded to the chimeric polypeptide by routine procedures.

One method of imaging the sites of inflammation in a patient involves detecting the expression of the L-selectin ligand on the inflamed endothelium. The method includes administering to a patient a pharmaceutical composition consisting of a detectable amount of a labeled ligand binding fragment of L-selectin, alone or joined to a carrier protein, including a ligand binding portion of a different selectin receptor, in a pharmaceutically acceptable carrier. Sufficient time is allowed for the labeled polypeptide to localize at the site of L-selectin ligand expression, unbound polypeptide is permitted to clear from healthy tissue in the patient, and signal generated by the label is detected and converted into an image of the site of inflammation. The amount of labeled ligand binding fragment of L-selectin preferably would be from 1 pg/kg to 10 μg/kg although higher or lower doses are possible depending on the imaging agent label used and the sensitivity of the detection method.

Some of the labels which can be detected externally from within the body of a human patient include radionuclides, radiopaque labels, and paramagnetic isotopes. A radionuclide for in vivo diagnosis should have a half-life long enough that it is still detectable at the time of maximum uptake, but short enough that after diagnosis unwanted radiation does not remain in the patient. Coupling of radionuclides to antibodies or proteins is well known in the art (see, e.g., Daddona et al., U.S. Pat. No. 5,026,537, the teachings of which are incorporated by reference herein) and is often accomplished either directly or indirectly using an intermediary linking group. Examples of radioisotopes that could be used for in vivo diagnosis are ⁹⁹ Tc, ¹²³ I, ¹³¹ I, ¹¹¹ In, ⁹⁷ Ru, ⁹⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹ Zr, and ²⁰¹ Ti. Paramagnetic isotopes for purposes of in vivo diagnosis can also be used according to the methods of this invention. Examples of elements that are particularly useful for use in magnetic resonance energy techniques include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶ Fe. For radiopaque imaging, the LAM-1 protein or ligand binding fragment is coupled to an agent that produces an opaque field at the site of inflammation upon X-ray imaging.

A ligand binding portion of the lectin or EGF-like domains of various selectins can be experimentally determined using this invention. In a typical procedure, fragments of the selectin cDNA can be fused with cDNA encoding a carrier protein, such as immunoglobulin heavy chain or another non-ligand binding portion of a different selectin. These chimeric proteins can be expressed on the cell surface, or as soluble molecules, and their ability to bind ligand assessed. Further segregated fragments of the lectin or EGF-like domain under study can be examined for their ability to bind ligands, as well as their ability to inhibit the binding of the parent selectin molecule to ligand.

Any of the peptides or polypeptides of the invention comprising lectin and EGF ligand binding portions from two different selectin molecules (including portions of any of the remaining domains (SCR, transmembrane or cytoplasic) of a generic selectin molecule or including a carrier protein) can be used to screen for antagonists of lectin or EGF function individually or for agents that can simultaneously antagonize the functions of the lectin and EGF domain of different selecting. This invention will allow the development of pharmacologic reagents composed of peptides, carbohydrate moieties, RNAs or other small molecules which may mimic the ligand of the lectin or EGF domains of the selectins or may mimic the ligand-binding epitopes of the respective selectin domains and thus be useful therapeutic agents.

In another therapeutic method, chimeric selectin polypeptides can be used in combination therapy with any other selectin or any other cell surface molecule, or soluble fragments thereof, involved in adhesion of leukocytes to endothelial surfaces. In addition, antagonists to any of the above receptors or receptor portions (or mAb reactive to the receptors or receptor portions) can be used in the above combinations or as independent antagonist combinations for treatment of a patient. Examples include ICAM-1, VCAM-1, VLA-4, CD18, CD11a, CD11b and CD31, and the mAb reactive with them.

The therapeutic agents may be administered orally, topically, or parenterally, (e.g., intranasally, subcutaneously, intramuscularly, intravenously, or intra-arterially) by routine methods in pharmaceutically acceptable inert carrier substances. Optimal dosage and modes of administration can readily be determined by conventional protocols.

The lyam-1 gene itself or portions thereof can be used to construct chimeric selectins. The gene can also be used in genetic therapy. Individuals having a genetic defect in the lyam-1 gene would be unable to produce a fully active L-selectin leukocyte "homing" receptor and thus would be unable to mobilize sufficient leukocytes to a site of inflammation. Individuals suspected of having a congenital defect in the lyam-1 gene could be screened for this genetic disorder using the sequence and structural information described. Treatment of affected individuals would then be possible using the lyam-1 gene or fragments thereof.

The normal regulation of the lyam-1 gene, as evidenced by the appearance and disappearance of L-selectin on the surface of a specific leukocyte sub-population can be monitored to test the effects of drugs or specific therapies that would alter gene expression.

While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 28                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2330 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 53..1207                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GAATTCCCTTTGGGCAAGGACCTGAGACCCTTGTGCTAAGTCAAGAGGCTCAATG55                      Met                                                                            GGCTGCAGAAGAACTAGAGAAGGACCAAGCAAAGCCATGATATTTCCA103                            GlyCysArgArgThrArgGluGlyProSerLysAlaMetIlePhePro                               51015                                                                          TGGAAATGTCAGAGCACCCAGAGGGACTTATGGAACATCTTCAAGTTG151                            TrpLysCysGlnSerThrGlnArgAspLeuTrpAsnIlePheLysLeu                               202530                                                                         TGGGGGTGGACAATGCTCTGTTGTGATTTCCTGGCACATCATGGAACC199                            TrpGlyTrpThrMetLeuCysCysAspPheLeuAlaHisHisGlyThr                               354045                                                                         GACTGCTGGACTTACCATTATTCTGAAAAACCCATGAACTGGCAAAGG247                            AspCysTrpThrTyrHisTyrSerGluLysProMetAsnTrpGlnArg                               50556065                                                                       GCTAGAAGATTCTGCCGAGACAATTACACAGATTTAGTTGCCATACAA295                            AlaArgArgPheCysArgAspAsnTyrThrAspLeuValAlaIleGln                               707580                                                                         AACAAGGCGGAAATTGAGTATCTGGAGAAGACTCTGCCTTTCAGTCGT343                            AsnLysAlaGluIleGluTyrLeuGluLysThrLeuProPheSerArg                               859095                                                                         TCTTACTACTGGATAGGAATCCGGAAGATAGGAGGAATATGGACGTGG391                            SerTyrTyrTrpIleGlyIleArgLysIleGlyGlyIleTrpThrTrp                               100105110                                                                      GTGGGAACCAACAAATCTCTCACTGAAGAAGCAGAGAACTGGGGAGAT439                            ValGlyThrAsnLysSerLeuThrGluGluAlaGluAsnTrpGlyAsp                               115120125                                                                      GGTGAGCCCAACAACAAGAAGAACAAGGAGGACTGCGTGGAGATCTAT487                            GlyGluProAsnAsnLysLysAsnLysGluAspCysValGluIleTyr                               130135140145                                                                   ATCAAGAGAAACAAAGATGCAGGCAAATGGAACGATGACGCCTGCCAC535                            IleLysArgAsnLysAspAlaGlyLysTrpAsnAspAspAlaCysHis                               150155160                                                                      AAACTAAAGGCAGCCCTCTGTTACACAGCTTCTTGCCAGCCCTGGTCA583                            LysLeuLysAlaAlaLeuCysTyrThrAlaSerCysGlnProTrpSer                               165170175                                                                      TGCAGTGGCCATGGAGAATGTGTAGAAATCATCAATAATTACACCTGC631                            CysSerGlyHisGlyGluCysValGluIleIleAsnAsnTyrThrCys                               180185190                                                                      AACTGTGATGTGGGGTACTATGGGCCCCAGTGTCAGTTTGTGATTCAG679                            AsnCysAspValGlyTyrTyrGlyProGlnCysGlnPheValIleGln                               195200205                                                                      TGTGAGCCTTTGGAGGCCCCAGAGCTGGGTACCATGGACTGTACTCAC727                            CysGluProLeuGluAlaProGluLeuGlyThrMetAspCysThrHis                               210215220225                                                                   CCTTTGGGAAACTTCAACTTCAACTCACAGTGTGCCTTCAGCTGCTCT775                            ProLeuGlyAsnPheAsnPheAsnSerGlnCysAlaPheSerCysSer                               230235240                                                                      GAAGGAACAAACTTAACTGGGATTGAAGAAACCACCTGTGAACCATTT823                            GluGlyThrAsnLeuThrGlyIleGluGluThrThrCysGluProPhe                               245250255                                                                      GGAAACTGGTCATCTCCAGAACCAACCTGTCAAGTGATTCAGTGTGAG871                            GlyAsnTrpSerSerProGluProThrCysGlnValIleGlnCysGlu                               260265270                                                                      CCTCTATCAGCACCAGATTTGGGGATCATGAACTGTAGCCATCCCCTG919                            ProLeuSerAlaProAspLeuGlyIleMetAsnCysSerHisProLeu                               275280285                                                                      GCCAGCTTCAGCTTTACCTCTGCATGTACCTTCATCTGCTCAGAAGGA967                            AlaSerPheSerPheThrSerAlaCysThrPheIleCysSerGluGly                               290295300305                                                                   ACTGAGTTAATTGGGAAGAAGAAAACCATTTGTGAATCATCTGGAATC1015                           ThrGluLeuIleGlyLysLysLysThrIleCysGluSerSerGlyIle                               310315320                                                                      TGGTCAAATCCTAGTCCAATATGTCAAAAATTGGACAAAAGTTTCTCA1063                           TrpSerAsnProSerProIleCysGlnLysLeuAspLysSerPheSer                               325330335                                                                      ATGATTAAGGAGGGTGATTATAACCCCCTCTTCATTCCAGTGGCAGTC1111                           MetIleLysGluGlyAspTyrAsnProLeuPheIleProValAlaVal                               340345350                                                                      ATGGTTACTGCATTCTCTGGGTTGGCATTTATCATTTGGCTGGCAAGG1159                           MetValThrAlaPheSerGlyLeuAlaPheIleIleTrpLeuAlaArg                               355360365                                                                      AGATTAAAAAAAGGCAAGAAATCCAAGAGAAGTATGAATGACCCATAT1207                           ArgLeuLysLysGlyLysLysSerLysArgSerMetAsnAspProTyr                               370375380385                                                                   TAAATCGCCCTTGGTGAAAGAAAATTCTTGGAATACTAAAAATCATGAGATCCTTTAAAT1267               CCTTCCATGAAACGTTTTGTGTGGTGGCACCTCCTACGTCAAACATGAAGTGTGTTTCCT1327               TCAGTGCATCTGGGAAGATTTCTACCTGACCAACAGTTCCTTCAGCTTCCATTTCACCCC1387               TCATTTATCCCTCAACCCCCAGCCCACAGGTGTTTATACAGCTCAGCTTTTTGTCTTTTC1447               TGAGGAGAAACAAATAAGACCATAAAGGGAAAGGATTCATGTGGAATATAAAGATGGCTG1507               ACTTTGCTCTTTCTTGACTCTTGTTTTCAGTTTCAATTCAGTGCTGTACTTGATGACAGA1567               CACTTCTAAATGAAGTGCAAATTTGATACATATGTGAATATGGACTCAGTTTTCTTGCAG1627               ATCAAATTTCGCGTCGTCTTCTGTATACGTGGAGGTACACTCTATGAAGTCAAAAGTCTA1687               CGCTCTCCTTTCTTTCTAACTCCAGTGAAGTAATGGGGTCCTGCTCAAGTTGAAAGAGTC1747               CTATTTGCACTGTAGCCTCGCCGTCTGTGAATTGGACCATCCTATTTAACTGGCTTCAGC1807               CTCCCCACCTTCTTCAGCCACCTCTCTTTTTCAGTTGGCTGACTTCCACACCTAGCATCT1867               CATGAGTGCCAAGCAAAAGGAGAGAAGAGAGAAATAGCCTGCGCTGTTTTTTAGTTTGGG1927               GGTTTTGCTGTTTCCTTTTATGAGACCCATTCCTATTTCTTATAGTCAATGTTTCTTTTA1987               TCACGATATTATTAGTAAGAAAACATCACTGAAATGCTAGCTGCAACTGACATCTCTTTG2047               ATGTCATATGGAAGAGTTAAAACAGGTGGAGAAATTCCTTGATTCACAATGAAATGCTCT2107               CCTTTCCCCTGCCCCCAGACCTTTTATCCACTTACCTAGATTCTACATATTCTTTAAATT2167               TCATCTCAGGCCTCCCTCAACCCCACCACTTCTTTTATAACTAGTCCTTTACTAATCCAA2227               CCCATGATGAGCTCCTCTTCCTGGCTTCTTACTGAAAGGTTACCCTGTAACATGCAATTT2287               TGCATTTGAATAAAGCCTGCTTTTTAAGTGTTAAAAAGAATTC2330                                (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 385 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetGlyCysArgArgThrArgGluGlyProSerLysAlaMetIlePhe                               151015                                                                         ProTrpLysCysGlnSerThrGlnArgAspLeuTrpAsnIlePheLys                               202530                                                                         LeuTrpGlyTrpThrMetLeuCysCysAspPheLeuAlaHisHisGly                               354045                                                                         ThrAspCysTrpThrTyrHisTyrSerGluLysProMetAsnTrpGln                               505560                                                                         ArgAlaArgArgPheCysArgAspAsnTyrThrAspLeuValAlaIle                               65707580                                                                       GlnAsnLysAlaGluIleGluTyrLeuGluLysThrLeuProPheSer                               859095                                                                         ArgSerTyrTyrTrpIleGlyIleArgLysIleGlyGlyIleTrpThr                               100105110                                                                      TrpValGlyThrAsnLysSerLeuThrGluGluAlaGluAsnTrpGly                               115120125                                                                      AspGlyGluProAsnAsnLysLysAsnLysGluAspCysValGluIle                               130135140                                                                      TyrIleLysArgAsnLysAspAlaGlyLysTrpAsnAspAspAlaCys                               145150155160                                                                   HisLysLeuLysAlaAlaLeuCysTyrThrAlaSerCysGlnProTrp                               165170175                                                                      SerCysSerGlyHisGlyGluCysValGluIleIleAsnAsnTyrThr                               180185190                                                                      CysAsnCysAspValGlyTyrTyrGlyProGlnCysGlnPheValIle                               195200205                                                                      GlnCysGluProLeuGluAlaProGluLeuGlyThrMetAspCysThr                               210215220                                                                      HisProLeuGlyAsnPheAsnPheAsnSerGlnCysAlaPheSerCys                               225230235240                                                                   SerGluGlyThrAsnLeuThrGlyIleGluGluThrThrCysGluPro                               245250255                                                                      PheGlyAsnTrpSerSerProGluProThrCysGlnValIleGlnCys                               260265270                                                                      GluProLeuSerAlaProAspLeuGlyIleMetAsnCysSerHisPro                               275280285                                                                      LeuAlaSerPheSerPheThrSerAlaCysThrPheIleCysSerGlu                               290295300                                                                      GlyThrGluLeuIleGlyLysLysLysThrIleCysGluSerSerGly                               305310315320                                                                   IleTrpSerAsnProSerProIleCysGlnLysLeuAspLysSerPhe                               325330335                                                                      SerMetIleLysGluGlyAspTyrAsnProLeuPheIleProValAla                               340345350                                                                      ValMetValThrAlaPheSerGlyLeuAlaPheIleIleTrpLeuAla                               355360365                                                                      ArgArgLeuLysLysGlyLysLysSerLysArgSerMetAsnAspPro                               370375380                                                                      Tyr                                                                            385                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1192 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        AGAGAGCTGTTATAAAGATTAAACAATATAATAAATATGGCGCGTGAGCTTCAGAGTTTT60                 TGTTGTTGTTATTATTATTTTCCTAAAAATGCAAATCTGATTTGCATTTGACTCATTGAC120                TCACATCAGTGGGTCTTCCTTTTTATTGTCCTTCATCATATGGGTCCTAATTTCACATGC180                AGTCTTATAAAACCATCTCATTTTATAGTCCAAGAATATTAAAGGTACTTGTAGGCTCCC240                AAACCTACACGGTGAAAAGCTAGAGAGCATGGGCTCTCTTCAGGGGTTAACTTCAGGAAG300                TGCCACTAACAAGGACGTCCACTAGGTGGTGAGCAAGGAAAGACGGAGGTGAAGGAACCG360                AAACGAGTCAAGTCCACTGCTTAGCTCTACTGAAGTTTTGCAAACATCATAAATATGTCT420                GAAATGCAGTTTTGATTTGTAGTATTTGCAATTTCCAAGGGCCATTTACCACAGGTAGCC480                AAGAGTTAGTTTAGCATTTATGAAAAAGATAGGGGAGGGTGGTGGTTAAGAAGGAGGTGG540                AGGAGAGAGTGAAGGAGGAAGAGGAGAACAAGAACCAAACAAAAACAAGAACAAGAACAA600                GTAGAAGAAGAGGAGCAGGGAGGAAAAAGAAGAGGAAGAAGAACAGCAACAACAATGAGT660                GAAGGAGGAGGAGGGTAAGGAAAGATGCATAGGAGAATGGAAGGAAGGATAGAAAGGAGG720                GAAGGAAGAGAGAATCTAGTCACATTACTTTCTGATCAGCAGTTCATTTTTGTCTCAGTG780                GGAGGCAATAGAGGCCAGTCTAGGAAAGGGGTGGGGAAAGAGGAAAGAGAAGTGCAGGAG840                GAAGGGGAGGCCCAAGGGGAGGAGGAGGAGGATGTGAGACTGGGTTAGAGAAATGAAAGA900                AAGCAAGGCTTTCTGTTGACATTCAGTGCAGTCTACCTGCAGCACAGCACACTCCCTTTG960                GGCAAGGACCTGAGACCCTTGTGCTAAGTCAAGAGGCTCAATGGGCTGCAGAAGAACTAG1020               AGAAGGACCAAGCAAAGCCATGGTGAGCCTTTCAGCCTAAAAGACGTTTAGATGCTCAGA1080               TAGAAACTCTTGGGGTTGTAGAGGCAGGTGGCAAGGATAGGAATCACCCCATTTCAATTC1140               TGGTTTTAAATAATATAGAAACTAAACATTTTCTCAGACCCTCAAAAAAAGT1192                       (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 363 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        CACTGAGACTAAGCGTAAAATAAATAGAACAAACAAACTGTGCATCAGTTCTGATGTAAA60                 TTTGAAGTAATTTTCATCTATGTCTGAGAAACCTGTTACCTCAGACAGGGTTAGTAGACA120                TATGTGTTTTATTCTGATTATTAAGAAAGTTGTAAGCACCACCTCAAAGGCTATAAATGT180                GTGGTTTAAGGGTATACATCTAAATATAATTTTGTATTTCATTTGCAGATATTTCCATGG240                AAATGTCAGAGCACCCAGAGGGACTTATGGAACATCTTCAAGTTGTGGGGGTGGACAATG300                CTCTGTTGTGGTATGTTATGATATTTATATATCACTAAGTCTATTTTACTTATATTCATT360                TTT363                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 531 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        CTGGAGTAGTGCTAGGTTCTTTTTAGCTGTAACATTATGTAAGTCTGCATAGGTCACACT60                 GATGTCTTGCAGATTTCCTGGCACATCATGGAACCGACTGCTGGACTTACCATTATTCTG120                AAAAACCCATGAACTGGCAAAGGGCTAGAAGATTCTGCCGAGACAATTACACAGATTTAG180                TTGCCATACAAAACAAGGCGGAAATTGAGTATCTGGAGAAGACTCTGCCTTTCAGTCGTT240                CTTACTACTGGATAGGAATCCGGAAGATAGGAGGAATATGGACGTGGGTGGGAACCAACA300                AATCTCTCACTGAAGAAGCAGAGAACTGGGGAGATGGTGAGCCCAACAACAAGAAGAACA360                AGGAGGACTGCGTGGAGATCTATATCAAGAGAAACAAAGATGCAGGCAAATGGAACGATG420                ACGCCTGCCACAAACTAAAGGCAGCCCTCTGTTACACAGGTAGGGAGTGACAAGACGGCT480                ATGCTGCCTCAGACTCAGGAAGGGCCACGGTTAAGAGAATACTCAGATTTA531                         (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 832 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        AAAATTTTAGCCATATGATTTTTATGCTATGAATTTACCAAATAAACCTTTCCTGATTAT60                 TTAAATCATCTCAGACAAAAGGTTATCTATGTCTAAAGAAATGACTTTGAGTACTAAAAT120                GTAATCACATTAAAATATTTTTTTTCTGACCTCCTTAAAGCTTCTTGCCAGCCCTGGTCA180                TGCAGTGGCCATGGAGAATGTGTAGAAATCATCAATAATTACACCTGCAACTGTGATGTG240                GGGTACTATGGGCCCCAGTGTCAGTTTGGTAAGTCTCTTTCCTTTCTTTGCTTCTTCTTA300                GGTAAAGTCACAGGAATCATTATAGCTTATCATGAAGCTGGTTGGAACAAAATGATACTA360                GCCACTCTGAGAAATGGGAAGTTTTGATCAGAAAGCTCTGCTTTCACAATATTGTTACCT420                TTCCGTAAAGATTTCATAAGTCAGCATGAAGTTTCGATTCACTTCTCAACAAGTCTTTTT480                GAGTACCACAAGAAGCACAGTGTTGGGATAAAGCTGTCAGGGTTACAATAAGGAATTAGC540                ATGGTAGATTCCCGCTCTCAAGAAGCTCACGATCTAATGAGCTTGTTAGATTAATTAGAA600                CTCTAAGGTCTGGAAGAAACTATGCCATTTATCATTAGGAGGCTGAGTTACCCAGAAAGT660                ATCTTGCTTTTTCCTTCTAGTAGTTCCTTTCCTTCTTGCAGTTCTCCACACTTAACACAT720                GTGCTCTGTAGCACACTGACTTTGCTGGTGGCCTTCTCTCTCATTTTGCACATGGCCAAA780                AAACATGTCATCTTTAAGACATTGTTCAAAGACAGTTTCTTCTAGGAAGCTT832                        (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 712 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        CTCTGATGTGATAGTTATTTCCCGACTAAGCTGGTCATTCCCAGTTACACCTATTTGGCT60                 TTAAGGATTCTCACTACAGATAATACTGAAGATAATAATATGAAGACTAGCTAATGTTTA120                CTTAGAATTTCTGATGAGTCAGGCTTTGTTCTAACGTCCTTGACTTATGCTAATTGAATT180                ACATTTAGTTTCCATATCAATTTGATAAAGATAACACAATTTCATTATTCCTCTTATATA240                GATGAAGAAACTGAAGTTGGAGGGTTCAAGTAACCTTGTTTAAAGGCACATGGTTATCAA300                GTGGCAGGGCTAGGATTCAAATCCAGGCGTCAGTTCCTCTTAACTCTTCCCCATACTGTT360                TCTTTCCCTATTGAAGTGATTCAGTGTGAGCCTTTGGAGGCCCCAGAGCTGGGTACCATG420                GACTGTACTCACCCTTTGGGAAACTTCAGCTTCAGCTCACAGTGTGCCTTCAGCTGCTCT480                GAAGGAACAAACTTAACTGGGATTGAAGAAACCACCTGTGGACCATTTGGAAACTGGTCA540                TCTCCAGAACCAACCTGTCAAGGTGAGTAACTTCAGACTAGAGGTTTTGTCATGCAATCC600                TGGGCTTACAGTCAGAACATTCAGTAGAAGTTTGCTGAGAAGTCAAACTTAGGATCCTAA660                TTTAACCTAACTTTTGTTTAACCTACTGTGATGTTTCTCAAAGGACTTATTC712                        (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 451 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        GAGGGTCACCTTAGCTAGGGCAGCAGCCTGGAGTAGCTACTCCTCTCCCCACAGCTTTCA60                 ATGCTTCCTTGCCTTCATCTCTCATTCACCACCCACCATCATTCTCAAGAAAATAAAGCC120                TGGAAGCAATATCACAAGTAATGTAGTCAGGCAGCTTTGGCTAAAAATCCAAAGCTCAAG180                GGAGGGTCTCTACTCAGAAATACTGTTTTGTCTTTTTTTTTTTTTCTTTTTCATTGAAGT240                GATTCAGTGTGAGCCTCTATCAGCACCAGATTTGGGGATCATGAACTGTAGCCATCCCCT300                GGCCAGCTTCAGCTTTACCTCTGCATGTACCTTCATCTGCTCAGAAGGAACTGAGTTAAT360                TGGGAAGAAGAAAACCATTTGTGAATCATCTGGAATCTGGTCAAATCCTAGTCCAATATG420                TCAAAGTGAGTAAGTTTGTCCTGGAACTGAA451                                             (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 544 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        TATCAGAACTAAGAAAGCTTGGGCTGCAGGTCGACTCTAGGTGCATTTTCAGGAACTCTA60                 TGAACCACAAATCTGGGCATTGAGATTCTGTAGGCATTAGACTAGCAAGGCTGGTCAGTC120                TTTGCCTATGCTGTAGACTCATCAGGGGCCTTCCCATGCCAGTTTCCTCATCTGTCAAAT180                GGCATCATTTGGGCTACTACTGGGAGATGTAAGGAGGAAAAAAGTCAAATATCATGAGAT240                AGACTAAGGAAATAATGCTGGTGGTCTCATGCTATGTGCCTTACTGATTTCTCTTTCAGA300                ATTGGACAAAAGTTTCTCAATGATTAAGGAGGGTGATTATAACCCCCTCTTCATTCCAGT360                GGCAGTCATGGTTACTGCATTCTCTGGGTTGGCATTTATCATTTGGCTGGCAAGGAGATT420                AAAAAAAGGTATGTGAGTTTAACTTCACATGAAAAGAACACAACTTTAAAGTGAAAAAGA480                AAAAAAAAAGAAACCCACAGGAAATTAAATGTGATAGATTCAACACAAGCAGGATGCCAA540                GCTT544                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 524 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       TAGTTTACAGTATTAGCAGCTGTCCCTCAAGGAAGAATCTGCAGGTAGATGAGATGCAGA60                 TTGGGTGGGATAAACACTTGAATGACATATTGGGTCTTGCCACCAGGCAATTTAGCAATT120                CTGTCTTCTTGAGTAGCACGGAGATGGAATGGAACCTCAGGAGGCATCTGCATCAACATG180                TCTGTTCTGTATTAGTGTCTACCACTGTTTATTAAGCCAGTTCCTCAAATCTCCTTTGAC240                ACAGATAGGGTCCACCTAACAAATACCTAATATACTTCAAAAGACAGTTTTGAGAGTGGG300                AGTCTTCCTTCTCCCTTACTTGAAAAACTTTAAATTGTCTAATTTTTGCTAATGCCTTTT360                TCTCTATTTTCTATTTCAGGCAAGAAATCCAAGAGAAGGTAAGTTTTATTAGTGGCGAGG420                AGTTTCCACATCTGCTGATTCATTCTCTACTTCTTAAGTTACTTCTGCTCTAGCTAGACA480                CATACCCATAGTAGTTATTACTGGGTCTATCAATGACAGATAGG524                                (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1696 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       ATAAGCATCACTAAAGAGCTTGTTAGGGGTGCAGAATCTCAGGCTCCACTCAGACCTACT60                 GAATCAGAGTCTGCATTTTAACACCATCTCTGAGTGGTAAGGACATGAAAATCTGAGAAG120                TGCTGCTACTAGGGTTTGCTTACATTTGTTCATCTTCAGAGGTTCCTAAAGCCTGGCCTC180                TTGTCTGAGATTTCCAGCTGAAAGCATTTCCTTGCTCCTCTTCTCATCTCTAATGAATAT240                TTACCTTTACTACTAACACTCCAAGTTTTGCAATTTTTAAACTCTTATTATCTTTTGTTT300                TTCTTTCAGTATGAATGACCCATATTAAATCGCCCTTGGTGAAAGAAAATTCTTGGAATA360                CTAAAAATCATGAGATCCTTTAAATCCTTCCATGAAACGTTTTGTGTGGTGGCACCTCCT420                ACGTCAAACATGAAGTGTGTTTCCTTCAGTGCATCTGGGAAGATTTCTACCTGACCAACA480                GTTCCTTCAGCTTCCATTTCACCCCTCATTTATCCCTCAACCCCCAGCCCACAGGTGTTT540                ATACAGCTCAGCTTTTTGTCTTTTCTGAGGAGAAACAAATAAGACCATAAAGGGAAAGGA600                TTCATGTGGAATATAAAGATGGCTGACTTTGCTCTTTCTTGACTCTTGTTTTCAGTTTCA660                ATTCAGTGCTGTACTTGATGACAGACACTTCTAAATGAAGTGCAAATTTGATACATATGT720                GAATATGGACTCAGTTTTCTTGCAGATCAAATTTCGCGTCGTCTTCTGTATACGTGGAGG780                TACACTCTATGAAGTCAAAAGTCTACGCTCTCCTTTCTTTCTAACTCCAGTGAAGTAATG840                GGGTCCTGCTCAAGTTGAAAGAGTCCTATTTGCACTGTAGCCTCGCCGTCTGTGAATTGG900                ACCATCCTATTTAACTGGCTTCAGCCTCCCCACCTTCTTCAGCCACCTCTCTTTTTCAGT960                TGGCTGACTTCCACACCTAGCATCTCATGAGTGCCAAGCAAAAGGAGAGAAGAGAGAAAT1020               AGCCTGCGCTGTTTTTTAGTTTGGGGGTTTTGCTGTTTCCTTTTATGAGACCCATTCCTA1080               TTTCTTATAGTCAATGTTTCTTTTATCACGATATTATTAGTAAGAAAACATCACTGAAAT1140               GCTAGCTGCAACTGACATCTCTTTGATGTCATATGGAAGAGTTAAAACAGGTGGAGAAAT1200               TCCTTGATTCACAATGAAATGCTCTCCTTTCCCCTGCCCCCAGACCTTTTATCCACTTAC1260               CTAGATTCTACATATTCTTTAAATTTCATCTCAGGCCTCCCTCAACCCCACCACTTCTTT1320               TATAACTAGTCCTTTACTAATCCAACCCATGATGAGCTCCTCTTCCTGGCTTCTTACTGA1380               AAGGTTACCCTGTAACATGCAATTTTGCATTTGAATAAAGCCTGCTTTTTAAGTGTTAAC1440               TAGTTTGCCTAGTTTGTTATTTTGAAAATTGATCATATGTTTTGTTTTCTCCCCAGTGAG1500               TTACATGCTCCTTCAGGGCAGAGTTTGTGTCAGATCCCTGGAGTATCTAGTGCATTACTT1560               GACACTCAATAAATGAATGTTCAAATAAATCAGAAAGAGCATACAGTGCACTGCTGATAT1620               AAGTTTCAGCATCCCTCTTTCTCTATGGCATCTGATGACCTGGGTCAGATATCACCTAAT1680               GTCAACAGCTGAATTC1696                                                           (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 119 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       TrpThrTyrHisTyrSerGluLysProMetAsnTrpGluAsnAlaArg                               151015                                                                         LysPheCysLysGlnAsnTyrThrAsnLeuValAlaIleGlnAsnLys                               202530                                                                         ArgGluIleGluTyrLeuGluAsnThrLeuProLysSerProTyrTyr                               354045                                                                         TyrTrpIleGlyIleArgLysIleGlyLysMetTrpThrTrpValGly                               505560                                                                         ThrAsnLysThrLeuThrLysGluAlaGluAsnTrpGlyAlaGlyGlu                               65707580                                                                       ProAsnAsnLysLysSerLysGluAspCysValGluIleTyrIleLys                               859095                                                                         ArgGluArgAspSerGlyLysTrpAsnAspAspAlaCysHisLysArg                               100105110                                                                      LysAlaAlaLeuCysTyrThr                                                          115                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 119 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       TrpThrTyrHisTyrSerGluArgSerMetAsnTrpGluAsnAlaArg                               151015                                                                         LysPheCysLysHisAsnTyrThrAspLeuValAlaIleGlnAsnLys                               202530                                                                         ArgGluIleGluTyrLeuGluLysThrLeuProLysAsnProThrTyr                               354045                                                                         TyrTrpIleGlyIleArgLysIleGlyLysThrTrpThrTrpValGly                               505560                                                                         ThrAsnLysThrLeuThrLysGluAlaGluAsnTrpGlyThrGlyGlu                               65707580                                                                       ProAsnAsnLysLysSerLysGluAspCysValGluIleTyrIleLys                               859095                                                                         ArgGluArgAspSerGlyLysTrpAsnAspAspAlaCysHisLysArg                               100105110                                                                      LysAlaAlaLeuCysTyrThr                                                          115                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 119 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       TrpThrTyrHisTyrSerLysArgProMetProTrpGluLysAlaArg                               151015                                                                         AlaPheCysArgGluAsnTyrThrAspLeuValAlaIleGlnAsnLys                               202530                                                                         GlyGluIleGluTyrLeuAsnLysThrGlnProPheSerArgThrTyr                               354045                                                                         TyrTrpIleGlyIleArgLysValGluGlyValTrpThrTrpValGly                               505560                                                                         ThrAsnLysSerLeuThrGluGluAlaLysAsnTrpGlyAlaGlyGlu                               65707580                                                                       ProAsnAsnArgLysSerLysGluAspCysValGluIleTyrIleLys                               859095                                                                         ArgAsnLysAspSerGlyLysTrpAsnAspAspAlaCysHisLysAla                               100105110                                                                      LysThrAlaLeuCysTyrThr                                                          115                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 119 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       TrpSerTyrAsnThrSerThrGluAlaMetThrTyrAspGluAlaSer                               151015                                                                         AlaTyrCysGlnGlnArgTyrThrHisLeuValAlaIleGlnAsnLys                               202530                                                                         GluGluIleGluTyrLeuAsnSerIleLeuSerTyrSerProSerTyr                               354045                                                                         TyrTrpIleGlyIleArgLysValAsnAsnValTrpValTrpValGly                               505560                                                                         ThrGlnLysProLeuThrGluGluAlaLysAsnTrpAlaProGlyGlu                               65707580                                                                       ProAsnAsnArgGlnLysAspGluAspCysValGluIleTyrIleLys                               859095                                                                         ArgGluLysAspValGlyMetTrpAsnAspGluArgCysSerLysLys                               100105110                                                                      LysLeuAlaLeuCysTyrThr                                                          115                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 119 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       TrpSerTyrHisAlaSerThrGluMetMetThrPheGluGluAlaArg                               151015                                                                         AspTyrCysGlnLysThrTyrThrAlaLeuValAlaIleGlnAsnGln                               202530                                                                         GluGluIleGluTyrLeuAsnSerThrPheSerTyrSerProSerTyr                               354045                                                                         TyrTrpIleGlyIleArgLysIleAsnGlyThrTrpThrTrpIleGly                               505560                                                                         ThrAsnLysSerLeuThrLysGluAlaThrAsnTrpAlaProGlyGlu                               65707580                                                                       ProAsnAsnLysGlnSerAspGluAspCysValGluIleTyrIleLys                               859095                                                                         ArgGluLysAspSerGlyLysTrpAsnAspGluLysCysThrLysGln                               100105110                                                                      LysLeuAlaLeuCysTyrLys                                                          115                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 119 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       TrpThrTyrHisTyrSerThrLysAlaTyrSerTrpAsnIleSerArg                               151015                                                                         LysTyrCysGlnAsnArgTyrThrAspLeuValAlaIleGlnAsnLys                               202530                                                                         AsnGluIleAspTyrLeuAsnLysValLeuProTyrTyrSerSerTyr                               354045                                                                         TyrTrpIleGlyIleArgLysAsnAsnLysThrTrpThrTrpValGly                               505560                                                                         ThrLysLysAlaLeuThrAsnGluAlaGluAsnTrpAlaAspAsnGlu                               65707580                                                                       ProAsnAsnLysArgAsnAsnGluAspCysValGluIleTyrIleLys                               859095                                                                         SerProSerAlaProGlyLysTrpAsnAspGluHisCysLeuLysLys                               100105110                                                                      LysHisAlaLeuCysTyrThr                                                          115                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 119 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       TrpThrTyrAsnTyrSerThrLysAlaTyrSerTrpAsnAsnSerArg                               151015                                                                         ValPheCysArgArgHisPheThrAspLeuValAlaIleGlnAsnLys                               202530                                                                         AsnGluIleAlaHisLeuAsnAspValIleProPhePheAsnSerTyr                               354045                                                                         TyrTrpIleGlyIleArgLysIleAsnAsnLysTrpThrTrpValGly                               505560                                                                         ThrAsnLysThrLeuThrGluGluAlaGluAsnTrpAlaAspAsnGlu                               65707580                                                                       ProAsnAsnLysLysAsnAsnGlnAspCysValGluIleTyrIleLys                               859095                                                                         SerAsnSerAlaProGlyLysTrpAsnAspGluProCysPheLysArg                               100105110                                                                      LysArgAlaLeuCysTyrThr                                                          115                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 119 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       TrpThrTyrHisTyrSerAsnLysThrTyrSerTrpAsnTyrSerArg                               151015                                                                         AlaPheCysGlnLysTyrTyrThrLysLeuValAlaIleGlnAsnLys                               202530                                                                         AsnGluIleAlaTyrLeuAsnGluThrIleProTyrTyrAsnSerTyr                               354045                                                                         TyrTrpIleGlyIleArgLysIleAsnAsnLysTrpThrTrpValGly                               505560                                                                         ThrLysLysThrLeuThrGluGluAlaGluAsnTrpAlaAspAsnGlu                               65707580                                                                       ProAsnAsnLysArgAsnAsnGlnAspCysValGluIleTyrIleLys                               859095                                                                         SerLeuSerAlaProGlyLysTrpAsnAspGluProCysTrpLysArg                               100105110                                                                      LysArgAlaLeuCysTyrArg                                                          115                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       AlaSerCysGlnProGlySerCysAsnGlyArgGlyGluCysValGlu                               151015                                                                         ThrIleAsnAsnHisThrCysIleCysAspAlaGlyTyrTyrGlyPro                               202530                                                                         GlnCysGlnTyr                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       AlaSerCysGlnProGluSerCysAsnArgHisGlyGluCysValGlu                               151015                                                                         ThrIleAsnAsnAsnThrCysIleCysAspProGlyTyrTyrGlyPro                               202530                                                                         GlnCysGlnTyr                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       AlaSerCysLysProTrpSerCysSerGlyHisGlyGlnCysValGlu                               151015                                                                         ValIleAsnAsnTyrThrCysAsnCysAspLeuGlyTyrTyrGlyPro                               202530                                                                         GluCysGlnPhe                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       AlaAlaCysThrAsnThrSerCysSerGlyHisGlyGluCysValGlu                               151015                                                                         ThrIleAsnAsnTyrThrCysLysCysAspProGlyPheSerGlyLeu                               202530                                                                         LysCysGluGln                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       AlaSerCysThrAsnAlaSerCysSerGlyHisGlyGluCysIleGlu                               151015                                                                         ThrIleAsnSerTyrThrCysLysCysHisProGlyPheLeuGlyPro                               202530                                                                         AsnCysGluGln                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       AlaAlaCysAsnProThrProCysGlySerHisGlyGluCysValGlu                               151015                                                                         IleIleAsnAsnTyrThrCysGlnCysHisProGlyPheLysGlyLeu                               202530                                                                         LysCysGluGln                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       AlaSerCysGlnAspMetSerCysSerLysGlnGlyGluCysLeuGlu                               151015                                                                         ThrIleGlyAsnTyrThrCysSerCysTyrProGlyPheTyrGlyPro                               202530                                                                         GluCysGluTyr                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       AlaSerCysGlnAspMetSerCysSerAsnGlnGlyLysCysIleGlu                               151015                                                                         ThrIleGlySerTyrThrCysSerCysTyrProGlyPheTyrGlyPro                               202530                                                                         GluCysGluTyr                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       AlaSerCysGlnAspMetSerCysSerLysGlnGlyGluCysIleGlu                               151015                                                                         ThrIleGlyAsnTyrThrCysSerCysTyrProGlyPheTyrGlyPro                               202530                                                                         GluCysGluTyr                                                                   35                                                                             __________________________________________________________________________ 

What is claimed is:
 1. An essentially purified polypeptide comprising the lectin domain of L-selectin, or a ligand-binding fragment thereof, followed by the EGF domain of E-selectin, or a ligand-binding fragment thereof.
 2. An essentially purified polypeptide comprising the lectin domain of E-selectin, or a ligand-binding fragment thereof, followed by the EGF domain of P-selectin, or a ligand-binding fragment thereof.
 3. An essentially purified polypeptide comprising the lectin domain of P-selectin, or a ligand-binding fragment thereof, followed by the EGF domain of E-selectin, or a ligand-binding fragment thereof.
 4. The polypeptide according to any one of claims 1-3, wherein the EGF domain is followed by a selectin short consensus repeat (SCR) domain and a selectin transmembrane domain.
 5. A DNA molecule comprising a sequence encoding the polypeptide according to claim
 1. 6. The polypeptide according to any one of claims 1-3, further comprising an SCR domain and a transmembrane domain from the same selectin as the lectin domain.
 7. The polypeptide according to claim 6, joined to a carrier protein.
 8. The polypeptide according to claim 6, which is detectably labelled.
 9. The polypeptide according to any one of claims 1-3, joined to a carrier protein.
 10. The polypeptide according to claim 9, wherein the carrier protein is an immunoglobulin heavy chain constant region, and wherein the polypeptide is joined to the N-terminus of the immunoglobulin heavy chain constant region.
 11. A DNA molecule comprising a sequence encoding the polypeptide according to claim
 2. 12. The polypeptide according to any one of claims 1-3, which is detectably labelled.
 13. The polypeptide according to claim 12, wherein the detectable label is selected from the group consisting of radionuclides, radiopaque labels and paramagnetic isotopes.
 14. A DNA molecule comprising a sequence encoding the polypeptide according to claim
 3. 